Stem cells are defined as cells that are capable of a differentiation into many other differentiated cell types. Embryonic stem cells are stem cells from embryos that are capable of differentiation into most, if not all, of the differentiated cell types of a mature body. Stem cells are referred to as pluripotent, which describes this capability of differentiating into many cell types. A category of pluripotent stem cell of high interest to the research community is the human embryonic stem cell, abbreviated herein as hES cell, which is an embryonic stem cell derived from a human embryonic source. hES cells are of great scientific interest because they are capable of indefinite proliferation in culture and are thus capable, at least in principle, of supplying cells and tissues for replacement of failing or defective human tissue. Methods to culture human embryonic stem cells offer the potential of unlimited amounts of human cells and tissues for use in a variety of therapeutic protocols to assist in human health. It is envisioned that in the future hES cells will be proliferated and directed to differentiate into specific lineages so as to develop differentiated cells or tissues that can be transplanted into human bodies for therapeutic purposes.
One of most significant features of hES cells is the attribute of being capable of self-renewal. By that it is meant that the hES cells are capable of proliferating into multiple progeny stem cells, each of which seems to have the full potential of its ancestor cell. In other words, the progeny are renewed to have all the developmental and proliferative capacity of the parental cell. This attribute, combined with the pluripotency, are the traits that make hES cells candidates for many potential uses, since, in theory, hES cells can be reproduced indefinitely and in large numbers and then induced to become any cell type in the human body. The attribute of ability to self-renew appears closely linked to the attribute of being undifferentiated in the sense that at least given present knowledge, only undifferentiated hES cells are capable of indefinite self-renewal and as soon as the cells differentiate, the attribute of self-renewal capability is lost. Since hES cells will spontaneously differentiate, care must be taken in culture conditions to maintain the cells in an undifferentiated state.
Among the factors that have so far been identified as successful in maintaining hES cells in long term culture in an undifferentiated state are the medium in which the cells are grown and the substrate on which they are grown. Much progress has been made in defining media, which can be formulated to include the activators of FGF and TGF-beta pathway and suppressors of BMP and WNT pathways, which have the effect of enhancing the cells self-renewal. Considerably less information is available on the role of substrates and cell-substrate adhesion in hES cell survival and growth.
In the original co-culture experiments, hES cells were plated on a gelatin-coated surface containing mouse embryonic fibroblasts (MEFs) or other feeder cells. Thomson et al., Science 282, 1145-1147 (1998). Upon plating, it was found that the hES cells do not grow on top of feeder cells, but instead tend to occupy the exposed gelatin-coated surface. As the hES cells proliferate, these feeder cells are “pushed away” by the growing ES cell colony. Imreh et al., Stem Cells and Development, 13, 337-343 (2004). This observation suggests that the gelatin-coated surface along with the secreted factors provide a sufficient platform for the attachment. It was also discovered that growth of cells on feeder layers can be avoided through the use of “conditioned medium” or CM, which is medium in which feeder cells have been cultured. However, culture of hES cells on simple gelatin-coated surfaces even in CM leads to rapid differentiation of the cells. Xu et al., Nat. Biotechnol. 19, 971-974 (2001). To date, growth of undifferentiated ES cells without exposure to feeder cells has been achieved on surfaces coated with lysed MEFs in a medium containing FGF and LIF, laminin in MEF-CM, fibronectin in the media containing LIF, bFGF and TGF-beta and Matrigel®-coated surface in various media conditions. (e.g., Xu, supra; Amit et al., Biol. Reprod. 70, 837-845 (2004); Hoffman & Carpenter, Nat. Biotechnol. 23, 699-708 (2005)). Matrigel® is a commercially produced extracellular matrix material. Interestingly, there have been other reports mentioning the failure of ES cell culture in the presence of MEF-CM on surfaces coated with fibronectin, laminin and Matrigel®. A recent review summarizing advances in hES cell culture techniques attributes this variability to multiple factors including variability in media formulations, variability in feeder types used for CM production, batch-to-batch variability of the attachment substrates or even variability between hES cell lines including origin, passage number, karyotypic stability and epigenetic status. (Hoffman, supra).
It is important not to neglect the role of substrate attachment for successful hES cell growth. Identifying defined hES cell growth conditions requires the identification of defined growth media and a defined hES cell attachment surface. Screening well-defined surfaces in an array format allows rapid identification of specific molecules that promote hES cell adhesion. Because of the relatively small amount of the materials (e.g., cells and media) required to screen for cell adhesion to a surface microarray, the screen can be easily repeated and performed in parallel for multiple ES cell types and media formulations. Thus, this strategy offers a low-cost and rapid means to find defined conditions and therefore tame the variability present in hES cell culture literature.
To date, several successful examples of multicomponent microarrays in cell-based screens have been reported. Lam and co-workers fabricated a peptide-array prepared by contact spotting of peptides onto glyoxylyl-functionalized glass slides. Falsey et al., Bioconjug. Chem. 12, 346-353 (2001). These arrays were used to identify peptides promoting adhesion of a specific cancer cell line. Bhatia and co-workers presented fabrication of a microarray presenting combinations of several proteins fabricated via contact printing onto acrylamide-coated glass slides. This array was used to screen for protein combinations that assist the differentiation of mouse ES cell into hepatocytes. Langer and coworkers fabricated an array of polymeric materials by spotting combinations of monomers onto a glass slide followed by in situ polymerization. Subsequently, they used a collection of cells obtained by trypsinization of embryonic bodies derived from hES cells and identified several polymers promoting cell adhesion and differentiation in the presence of retinoic acid. A similar approach was recently reported to screen for materials promoting adhesion of the mesenchymal stem cells. However, no reports to date have described screening for growth of undifferentiated hES cells. Identifying such conditions is challenging: The undifferentiated state of hES cells is easily disrupted by small changes in their growth environment, an attribute that has hindered the development of a reliable and reproducible assay to assess a variety of growth conditions.